The goal of this project is to determine the role that costimulators expressed on B-cells play in the regulation of antibody responses to T- dependent antigens. Based in part on the observation that transgenic mice that constitutively express the costimulator B7-1 on all B-cells exhibit a profound defect in T-dependent antibody responses, we have proposed the novel hypothesis that costimulators expressed by B-cells can down-regulate as well as stimulate humoral responses to T-dependent antigens. The specific aims of the project are as follows: 1. Definition of the kinetics and anatomy of expression of costimulators and their counterreceptors during T-B interactions in vivo. Assessment of the functional roles of costimulators has been impeded by our limited knowledge of how different costimulators and their counter-receptors are expressed during T-B interactions in vivo. In order to address this issue, we will set up in vitro and in vivo systems using antigen-specific normal or Ig transgenic B-cells and T-cell receptor transgenic T-cells, in which antigen-specific T-B interactions can be directly assessed. Functional and morphologic assays will be used to define the kinetics and patterns of expression of costimulators and their counter-receptors on the interacting cell populations. 2. Role of costimulators in cognate T-B interactions. Based on the kinetics and anatomy of costimulator expression, as defined above, we will administer specific blocking antibodies to immunized mice to test the opposing hypotheses that a) different costimulators regulate T-dependent B- cell responses in distinct ways, and b) temporal expression patterns of costimulators are important determinants of their functions. The mechanisms by which dysregulated expression of one costimulator, B7-1, inhibits antibody responses will be defined, with specific reference to effects on helper T cell functions and survival and expansion of B cells. 3. Definition of the role of costimulators in vivo, as revealed in transgenic and knockout mice. In order to test the hypothesis that different costimulators serve distinct functions, we will generate and study B7-2 transgenic mice. The failure of B7-2 transgenic mice to respond to T-dependent antigens would support the hypothesis that B7-2 transgenic mice. The failure of B7-1 and B7-2 deliver similar signals. Conversely, if B7-2 transgenics exhibit a different phenotype, this would suggest that these two costimulators deliver fundamentally different signals. We will also reconstitute irradiated wild-type mice with B-cells from B7-1 or B7-2 knockout mice, in order to study the effects of isolated B cell costimulator deficiency.